Fig. 6

HNF4α regulates HOTAIR expression in colon cells. a RT-qPCR analysis for the indicated markers on SW480 cells overexpressing HNF4α (HNF4α), compared with mock-infected control cells (Ctr). The values are calculated by the ΔCt method, expressed as fold of expression vs. the control (arbitrary value = 1) and shown as means ± S.E.M. Statistically significant differences are reported. Statistically significant differences (*p < 0.05; **p < 0.01; ***p < 0.001) are reported for five independent experiments. b Western blot analysis for the indicated markers on protein extracts from cells treated as in a. Protein amount was normalized by immunoblotting for GAPDH, as indicated. All the experiments have been performed in triplicate and WB images represent one indicative experiment of three independent ones. c RT-qPCR analysis as in a on HNF4α-silenced-SW620 cells (siHNF4α) in comparison with siGFP cells (siCtr), as control. Statistically significant differences (*p < 0.05; **p < 0.01) are reported for four independent experiments. d qPCR analysis of ChIP assays with anti-HNF4α antibody (IP HNF4α) and, as control, normal rabbit IgG (IgG) on chromatin from SW480 and SW620 cells. Data show the direct recruitment of endogenous HNF4α on the correspondent consensus binding site on the human promoter of HOTAIR. Rpl30 promoter was used as a negative control. Values derived from five independent experiments are reported as means ± S.E.M. and expressed as percentage of the Input chromatin (% Input). Statistically significant differences (*p < 0.05; n.s. no significant) are reported